Translation, the process of protein synthesis, follows a universally conserved mechanism that is central to gene expression. Translation is highly regulated in human cells and loss of translation control is a key determinant of cancerous cell growth. Bacterial translation is targeted by a broad array of clinically-important therapeutic compounds that are used to combat infectious disease. However, resistance to known antibiotics is increasingly widespread. The ribosome is the principal component of the cellular translation apparatus and is the integration point for regulation.
The molecular mechanisms of ribosome regulation and antibiotic action remain poorly understood. To catalyze protein synthesis, the ribosome, a highly-conserved, RNA-protein assembly, works in concert with numerous RNA ligands and protein translation factors to convert a messenger RNA (mRNA) template into a specific polypeptide sequence. The mechanism of protein synthesis hinges on repetitive processes central to which is the incorporation of specific, aminoacylated transfer RNA (aa-tRNA) molecules for each mRNA codon, followed by movement of the ribosome along mRNA in discrete, codon steps. As we have demonstrated through our previous research, transient factor binding events and conformational changes in the ribosome are critical to the mechanism of this directional, high-fidelity process.
Messenger RNA (mRNA)-directed protein synthesis takes place on the two-subunit ˜2.4 MDa ribosome particle (70S in bacteria) during the elongation phase of translation. In this process, the ˜25 KDa, L-shaped aminoacyl-transfer RNA (aa-tRNA) molecule binds the ribosome within the aminoacyl (A) site through base pairing interactions with the mRNA codon within the small subunit (30S) decoding site (Ramakrishnan 2002; Yusupov 2001). The ribosome's recognition of the helical geometry arising from the paired mRNA codon and tRNA anticodon stimulates conformational events in the particle and tRNA that facilitate delivery of the amino acid linked to the distal 3′-CCA terminus of aa-tRNA into the peptidyltransferase center (PTC) of the large subunit (50S) more than 80 Å away (Ramakrishnan 2002; Ogle 2003; Schuette 2009; Villa 2009; Schmeing 2009a, 2009b; Rodnina 2009). The selection process terminates with peptide bond formation catalyzed by elements of the PTC. The mechanism of aa-tRNA selection establishes the genetic code by ensuring that correct (cognate) aa-tRNA are rapidly incorporated into the ribosome while near- and non-cognate aa-tRNAs are rapidly and efficiently rejected (Parker 1989; Rodnina 2005). In vivo and in vitro measurements estimate the rate of translation at ˜2-20 amino acids per second with error frequencies ranging from ˜1×10−2-10−6 depending on experimental conditions (Parker 1989; Johansson 2008; Ninio 2006).
A deeper understanding of the nature, role and timing of factor and tRNA binding events on the ribosome, as well as conformational events within components of the system underpinning rate-determining steps in the process are necessary to gain insight into translation regulation, and for drug screening assays aimed at targeting the translation apparatus for the therapeutic treatment of infection and cancer. Historically, bulk biochemical and biophysical experiments have been employed with the goal of obtaining such information. However, such efforts are hampered by the asynchronous nature of translation events and ensemble averaging phenomena that ultimately compromise quantitative analyses and interpretations. Studies performed in bulk also have been hampered by the need for milligram quantities of pure, highly-active components, which can really only be achieved for relatively simple, non-pathogenic strains of Escherichia coli (E. coli). As a result, a paucity of quantitative information is available for processive translation reactions for even the most simple, model organisms (e.g., E. coli) and almost no information whatsoever regarding the human translation apparatus—from which to advance therapeutic approaches to cancer treatment focused on targeting the translation machinery.
Recent advances in methods that enable the direct imaging of translation reactions using single-molecule fluorescence and fluorescence resonance energy transfer (FRET) overcome many of the aforementioned challenges. However, investigations of single-round and processive translation reactions can be hampered by the need to perform such experiments using relatively high concentrations of fluorescently labeled translation components in solution. Elevated concentrations of fluorescent components in solution cause unwanted background and a global deterioration of signal-to-noise ratios for the types of imaging platforms/strategies presently available including, for example, total internal reflection microscopy, confocal imaging and zero-mode waveguide directed imaging platforms such as those offered by Pacific Biosciences (Menlo Park, Calif.). Such considerations have proven to be a bottleneck for this important area of research and to overcome this limitation, the imaging of protein synthesis reactions must typically be performed using relatively low concentrations of fluorescently labeled translation components (1-200 nM). Such low concentrations hamper translation rates and lead to undesirable side reactions and fluorophore photobleaching, ultimately limiting the utility and interpretation of the measurements. Even the use of zero-mode waveguides, designed specifically to enable the use of higher concentrations of fluorescent components in solution, have failed to effectively overcome this obstacle.
In previous work, we aimed to diminish the negative consequences of unwanted fluorophore photobleaching through the design and synthesis of long-lasting and non-blinking fluorescent dye molecules. However, such compounds do not overcome the issues of background introduced by the use of high concentrations of fluorescent species needed for imaging experiments. Thus, forward progress in the field requires solutions that specifically address the need to use high concentrations of fluorescent agents in solution.
The present invention builds on previous technologies employed most visibly in the field of molecular beacons, which have been used to overcome similar hurdles in the imaging of biological systems using fluorescent probes. Molecular beacons are single-stranded oligonucleotide hybridization probes that form a stem-and-loop structure with a fluorophore covalently linked to the end of one arm and a quencher covalently linked to the end of the other arm. In the absence of targets, the probe is dark, because the stem places the fluorophore so close to the non-fluorescent quencher that they transiently share electrons, eliminating the ability of the fluorophore to fluoresce. When the probe encounters a target molecule, it forms a probe-target hybrid that is longer and more stable than the stem hybrid. Such technology has engendered success in numerous applications, including the in vivo imaging of specific mRNA templates. Proximal quenching hinges on the transfer of energy through space from an excited (illuminated) fluorophore to a neighboring fluorophore that dissipates energy through non-fluorescing pathways (e.g. heat dissipation).
The present invention takes analogous approach to image translation events based on the principle that aminoacyl-tRNA (aa-tRNA) substrates are delivered to the ribosome in a “ternary complex” of elongation factor Tu (EF-Tu), aa-tRNA and GTP, which with labeling in accordance with the present invention, provides “dark” ternary complexes that only fluoresce upon delivery of the charged tRNA to the ribosome and dissociation of the ternary complex.
In previous research, fluorescently labeled tRNA was used to directly monitor translation reactions and drug activity on the ribosome. In the present invention, we describe methods developed to enable attachment of “quencher” fluorophores directly to EF-Tu in a manner that efficiently quenches the fluorophore linked to tRNA when they are bound together in a ternary complex by virtue of the fluorophore and quencher proximity, without compromising the biological activity of the ternary complex in any detectable manner. While originally developed as a means to monitor FRET within ternary complex and conformation changes therein during the delivery of aa-tRNA to the ribosome, we have since simplified this approach on the basis of efforts that enable the direct monitoring of the sequential tRNA incorporation reactions.
No fluorescence-based methods or assays to monitor the kinetic parameters of ternary complex formation and/or dissociation are currently available. In 1985, there was a report of a fluorescence-based assay for ternary complex formation using tRNAPhe labeled at the naturally occurring 4-thiouriding residue at position 8 of tRNA (Abrahamson 1985). However, this assay suffers from relatively poor activity of the components. In particular, the use of tRNAPhe labeled at the 4-thiouridine residue at position 8 suffers from reduced rates of aminoacylation which precludes robust kinetic studies of the process. It also does not employ fluorescently-labeled or quencher-labeled EF-Tu species. In 2010, there was a report published claiming a method for detecting FRET between EF-Tu and tRNA for purposes related to the detection of translation (Perla-Kajan 2010). However, the approach described the introduction of mutations in EF-Tu and a labeling approach that rendered EF-Tu all but inactive for ternary complex formation.
The present invention is unique as it employs novel derivatives of EF-Tu and tRNA where both species can be quantitatively labeled while remaining fully active in translation.